A Combination of Culture Conditions and Gene Expression Analysis Can Be Used to Investigate and Predict hES Cell Differentiation Potential towards Male Gonadal Cells

Human embryonic stem cell differentiation towards various cell types belonging to ecto-, endo- and mesodermal cell lineages has been demonstrated, with high efficiency rates using standardized differentiation protocols. However, germ cell differentiation from human embryonic stem cells has been very inefficient so far. Even though the influence of various growth factors has been evaluated, the gene expression of different cell lines in relation to their differentiation potential has not yet been extensively examined. In this study, the potential of three male human embryonic stem cell lines to differentiate towards male gonadal cells was explored by analysing their gene expression profiles. The human embryonic stem cell lines were cultured for 14 days as monolayers on supporting human foreskin fibroblasts or as spheres in suspension, and were differentiated using BMP7, or spontaneous differentiation by omitting exogenous FGF2. TLDA analysis revealed that in the undifferentiated state, these cell lines have diverse mRNA profiles and exhibit significantly different potentials for differentiation towards the cell types present in the male gonads. This potential was associated with important factors directing the fate of the male primordial germ cells in vivo to form gonocytes, such as SOX17 or genes involved in the NODAL/ACTIVIN pathway, for example. Stimulation with BMP7 in suspension culture resulted in up-regulation of cytoplasmic SOX9 protein expression in all three lines. The observation that human embryonic stem cells differentiate towards germ and somatic cells after spontaneous and BMP7-induced stimulation in suspension emphasizes the important role of somatic cells in germ cell differentiation in vitro.

已有研究证实，采用标准化分化方案，人类胚胎干细胞可高效分化为外胚层、内胚层及中胚层细胞谱系中的各类细胞。然而迄今为止，人类胚胎干细胞向生殖细胞的分化效率始终极低。尽管已有研究对多种生长因子的影响进行了评估，但目前尚未针对不同细胞系的基因表达及其分化潜能的关联展开广泛研究。本研究通过分析3株男性人类胚胎干细胞系的基因表达谱，探究了其向男性性腺细胞分化的潜能。本研究中将上述干细胞系以单层形式接种于人包皮成纤维细胞滋养层，或悬浮培养为细胞球，分别采用骨形态发生蛋白7（BMP7）诱导分化，或通过撤除外源性成纤维细胞生长因子2（FGF2）进行自发分化，培养时长均为14天。通过TaqMan低密度阵列（TLDA）分析发现，在未分化状态下，这3株细胞系的mRNA表达谱存在显著差异，且向男性性腺细胞的分化潜能亦存在显著不同。例如，该分化潜能与体内调控雄性原始生殖细胞发育为性原细胞的关键因子（如SOX17）以及参与NODAL/ACTIVIN信号通路的基因密切相关。悬浮培养中经BMP7刺激后，3株细胞系的胞质SOX9蛋白表达均显著上调。本研究观察到，人类胚胎干细胞经悬浮培养下的自发分化或BMP7诱导后，可向生殖细胞与体细胞分化，这一结果凸显了体细胞在体外生殖细胞分化过程中的关键作用。